Real-time polymerase chain reaction for the detection of toxigenic clostridium botulinum type C1 in waterbird and sediment samples: Comparison with other PCR techniques

A quantitative real-time PCR (qPCR) for detection of the neurotoxin of the Clostridium botulinum type C (BoNTC) encoding gene has been compared with a nested PCR (nPCR) and a conventional PCR (cPCR) using 2 toxigenic C. botulinum C1 reference strains and samples from bird tissues (n = 30) and sediments (n = 30) from wetlands where botulism outbreaks have been reported. A cPCR based on 16S ribosomal RNA sequences from 60 strains of Clostridium species was also developed to detect the genomic DNA of C. botulinum C in order to evaluate the presence of nontoxigenic strains. Quantitative PCR showed a similar sensitivity to nPCR (<0.5 pg of DNA), and both were more sensitive than the cPCR when using the C. botulinum reference strains. Quantitative PCR and nPCR revealed an equal number of positives in uncultured samples of sediments (3%) and bird tissues (40%), but these values tended to be higher after culture enrichment with the qPCR assay (10% and 80%, respectively). Associations between the presences of toxigenic C. botulinum C in the environment and in birds within the ecological conditions in wetlands could be studied further using the culture enrichment and qPCR techniques shown in the current study.